Dopamine (DA), acting through D1-like and D2-like receptors, has a major role in regulating neuronal motor control, cognition, event prediction and emotion (5-10). In mammals, five distinct genes, termed D1/D5 for D1-like receptors and D2/D3/D4 for D2-like receptors, encode DA receptors. These receptors belong to a super-family of single polypeptide 7-transmembrane domain receptors that exert their biological effects via intracellular G-protein coupled signalling cascades (11). D1 and D5 receptors preferentially couple to Gs proteins, stimulating the activity of adenylate cyclase and PKA dependent pathways. Dopamine D2 receptors (D2R) display a more complex pattern of signal transduction primarily due to their coupling to subtype-specific members of the Gi/Go protein family (11). D2R are known to stimulate a number of signal transduction pathways including the inhibition of adenylate cyclase activity, PI turnover, potentiation of arachidonic acid release, inwardly rectifying K+ and Ca2+ channels and mitogen activated protein kinases (12). D1- and D2-like receptors are further differentiated on pharmacological grounds with D1-like receptors selectively binding agonists [e.g. fenoldopam, SKF-81297] and antagonists [SCH-23390] of the benzazepine and benzonapthazine class with high affinity, while D2-like receptors bind selectively to wide variety of agonists and antagonists from numerous structural classes, including aminotetralins, butyrophenones and substituted benzamides. Furthermore, as opposed to the D1/D5 receptor genes that are intron-less, the molecular diversity and multiplicity of the D2-like receptor subfamily arises from alternative splicing of the D2R, termed D2Long (D2L) and D2Short (D2S) (12); abnormally spliced truncated variants of the D3 receptor (13-14) and numerous polymorphic variants of D4 receptors (15) as well as 19 additional allelic variants found in humans (16-17). The molecular isoforms of the D2R are identical except for the presence of a 29 amino acid insert in D2L while polymorphic D4 receptors differ in both the number and order of a 48 base-pair repeat sequence. These insertions/variations occur within the third cytoplasmic loop of D2/D4, a domain thought to encode sequence motifs enabling receptor coupling to specific G-proteins (18-19). In addition, several studies suggest that the D2S splice variant is predominantly found on pre-synaptic terminals while the D2L is mostly found post-synaptically (20-25).
As described above, D2R has been shown to regulate cAMP-PKA and Ca2+ pathways through Gi/o-dependent signaling (11, 26). However, recent studies have suggested that D2R activate the Akt/GSK-3 pathway via G protein-independent (β-arrestin 2-dependent) signaling (27, 28, 101-104). D2R-mediated Akt/GSK-3 regulation involves the recruitment of β-arrestin 2 to the D2R and the formation of signalling complexes containing β-arrestin 2, PP2A (protein phosphatase 2A), and Akt, which leads to specific dephosphorylation/inactivation of the serine/threonine kinase Akt on its regulatory Thr308 residue but not the second regulatory residue (Ser473) (28), Dephosphorylation of Akt in response to DA leads to a reduction of kinase activity and a concomitant activation of its substrates GSK-3α/β since both are negatively regulated by Akt (28-29). Functionally, pharmacological activation of Akt or inhibition of GSK3α/β results in reduction of DA-associated locomotor activity in both DAT-Knock-out mice and wild-type mice treated with amphetamine (28,30). Moreover, mice lacking one allele of the GSK-3β gene show markedly reduced locomotor responses to amphetamine (28), while mice lacking the Akt isoform Akt1 display behavioural manifestations generally associated with enhanced dopaminergic responses (31), thus supporting a role for the Akt/GSK3 signalling pathway in the expression of DA-associated behaviours. β-arrestin 2 deficiency in mice results in reduction of dopamine-dependent behaviours, loss of Akt regulation by DA in the striatum, and disruption of the DA-dependent interaction of Akt with its negative regulator, PP2A-indicating an important role for β-arrestin 2 in this process (27-28,32). Importantly, antipsychotics including haloperidol, clozapine, aripiprazole, chlorpromazine, quetiapine, olanzapine, risperidone, and ziprasidone all potently antagonize recruitment of β-arrestin 2 to D2R induced by activation of D2R (33-34). Lithium—a drug prescribed for the primary treatment of bipolar disorder, and used to augment treatment for schizophrenia and depression—regulates Akt/GSK3 signalling and related behaviours in mice by disrupting a signalling complex composed of Akt, β-arrestin 2, and PP2A (28,101). These data support an important role of D2R-mediated Akt/GSK-3 signalling in the pathology of mental illnesses.
Many studies demonstrate that D2R are involved in schizophrenia and antipsychotic medication action. First, there is a positive correlation between the clinical potency and D2R binding affinity of antipsychotic drugs (35-37). Second, there are increased levels of brain D2R in patients with schizophrenia as shown in post-mortem, PET and SPECT studies (38-44). Third, there are elevated D2R mRNA levels in the post-mortem frontal cortex of schizophrenia patients (45). Clinically, all current antipsychotics exert their effect through D2R3, and currently, there are no antipsychotics with a novel mechanism of action (46). The only new possibly different antipsychotic is the glutamate agonist LY404,039, which has been significantly effective in 100 Russian patients with schizophrenia (47).
In recent years, it has become evident that the diverse cellular properties of D2R can be regulated through their interaction with a class of molecules collectively termed DA receptor interacting proteins (DRIPs). DRIPs not only regulate receptor signalling, but contribute to receptor trafficking and stability. Proteins that interact with D2R include neurotransmitter receptors, transporters, ion channels, intracellular signalling proteins, cytoskeleton proteins, protein kinases and adaptor/chaperone proteins (48-67). Given that each specific protein-protein interaction enables the D2R to perform a specific function, the identification of D2R-interacting proteins may improve knowledge about the etiology of neuropsychiatric diseases and to develop treatments targeted at the underlying pathophysiology.
Disrupted-in-schizophrenia-1 (DISC1) was originally identified as a susceptibility gene for schizophrenia in a Scottish family carrying a balanced chromosomal translocation (1q42.1:11q14.3) that co-segregates with major mental illnesses including schizophrenia, bipolar disorder and major depression (LOD score 7.1) (68-70). Translocation carriers also showed a significant reduction in the P300 event-related potential-a general biomarker in schizophrenic patients (69). Genetic studies demonstrate significant linkage between the DISC1 locus and psychiatric illness in Finnish (71), Taiwanese (72) and Icelandic populations (73). Genetic association studies also support that DISC1 variants affect susceptibility to psychiatric disease (74-77). However, as with most complex disease phenotypes, some studies have not replicated these results, such as one with subjects of Japanese background (78).
DISC1 is comprised of 13 exons and encodes a protein of 854 amino acids that is conserved across primates and rodents, but shows little homology to other proteins and species (68, 79-80). Much remains unknown about DISC1 function, but the amino acid sequence suggests that it is likely to act as a scaffolding protein with multiple to binding motifs (81). The globular N-terminus contains nuclear localization signals. The coiled-coil C-terminus consists of different domains that allows DISC1 protein to interact with a variety of functionally diverse proteins in the brain, including: (a) microtubule-associated, centrosomal proteins including NudE-like (NUDEL), kendrin, microtubule-interacting protein associated with TRAF3 (MIPT3), and microtubule-associated protein 1A (MAP1A); (b) possible nuclear proteins such as activating transcription factor 4/5 (ATF4/5); (c) actin-associated proteins including spectrin and fasciculation and elongation protein zeta-1 (FEZ1); and (d) postsynaptic density-associated proteins that function in synaptic morphology and plasticity, such as Citron (82). Recently, phosphodiesterase 4B (PDE4B) was reported to bind to the NT of DISC1 (83). This interaction is predicted to play a regulatory role in cAMP signaling (80). The translocation results in a CT truncation of the DISC1 protein and thus affects its ability to form protein complexes and mediate downstream signals. Biochemical studies have indicated that DISC1 protein contains a self-interacting domain that allows the formation of a dimer. The truncated protein is thought to form a dimer with the wild-type protein, hence disturbing its normal function and subcellular distribution (84-85). This dominant-negative hypothesis was further supported by similar cellular effects observed with both the introduction of the CT-truncated mutant and suppression of endogenous DISC1 with RNA interference (84).
There are several types of genetic mouse models for DISC1, beginning with a spontaneous deletion variant in the DISC1 gene specific to the 129S6/SvEv strain (86). This mutation displays impaired working memory, which is consistent with the cognitive impairment in schizophrenia (86). Hikida et al. generated mice expressing a CT truncated DISC1 thought to act in a dominant negative fashion (DN-DISC1) and found significant anatomical (enlargement of lateral ventricles) and behavioural abnormalities (hyperactivity, disturbance in prepulse inhibition and depression-like deficits) (87). Interestingly, transgenic mice expressing only the DISC1 CT fragment (DISC1-cc) also resulted in schizophrenia-related phenotypes (88). Finally, Pletnikov's group derived transgenic mice with predominant expression of mutant human DISC1 (107). Overlapping anatomical and behavioural deficits such as enlarged lateral ventricles, neurite outgrowth defects, hyperactivity and abnormal spatial learning and memory functions were observed with this transgenic mouse line (89).
Two other independent mouse lines with DISC1 amino acid changes Q31L (127A/T) and L100P (334T/C)90 demonstrated both anatomical and behavioural changes consistent with those seen in schizophrenia: enlarged lateral ventricles on MRI, abnormal sensory gating measured with prepulse inhibition (PPI), deficits in anxiety-related exploratory behaviour and decreased social interaction. These abnormalities responded to the appropriate drug treatment used to target the same domains of dysfunction in humans. Each of the two mutant lines have overlapping but different types of behavioural deficits and pharmacological responses; the Q31L line has more depression-related symptoms whereas the L100P shows more psychosis-related symptoms (90). This is intriguing given the observation that the Scottish family members with the 1:11 translocation all shared the same breakpoint, but had a spectrum of clinical syndromes (68). These data point to new pathophysiological mechanisms involving dysfunction of DISC1 and GSK-3, and provide a link to the well-established DA hypothesis in schizophrenia.
There is a need in the art to identify novel therapeutic targets for treating schizophrenia and other medical conditions involving aberrant dopamine signalling and regulation. Further, there is a need in the art to identify new therapeutic agents for treating schizophrenia and other medical conditions involving aberrant dopamine signaling and regulation. There is also a need in the art for novel assays to identify such therapeutic agents.